Reactive & Functional Polymers 72 (2012) 107–113

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Reactive & Functional Polymers

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New method for the synthesis and purification of branched mPEG2lys

Marianela González, Ricardo J.A. Grau 1, Santiago E. Vaillard ⇑Laboratorio de Química Fina, INTEC (UNL-CONICET), Predio CCT-CONICET Santa Fe, Colect. Ruta Nacional 168, Km 472, Paraje ‘‘El Pozo’’, 3000 Santa Fe, Argentina

a r t i c l e i n f o

Article history:Received 9 May 2011Received in revised form 17 October 2011Accepted 22 October 2011Available online 15 November 2011

Keywords:Branched PEGReactive PEGPegylationImidazolium saltPolyethylene glycol

1381-5148/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.reactfunctpolym.2011.10.010

⇑ Corresponding author. Tel.: +54 342 4511370x11E-mail addresses: mgonzal@intec.unl.edu.ar (M.

conicet.gov.ar (R.J.A. Grau), svaillard@intec.unl.edu.ar1 Deceased.

a b s t r a c t

Branched PEG structures are particularly useful for the pegylation of different biomaterials. In this regard,the branched structure bearing a lysine core and two mPEG chains linked by urethane bonds (mPEG2lys)has been used for the pegylation of several important biomolecules. In this work we describe the prep-aration of a new functionalized mPEG featuring an alkoxy-carbonylimidazolium iodide reactive groupthat can be used for the urethane bonds formation required in mPEG2lys (20 kDa). The procedure forthe preparation of the mPEG-alkoxy-carbonylimidazolium iodide involves the initial reaction of mPEGwith N,N0-carbonylbismidazole (CDI) as phosgene equivalent, followed by alkylation with methyl iodide.The functionalized mPEG was reacted with a silylated lysine derivative affording mPEG2lys in a singlestep. Therefore, harmful reagents (like phosgene derivatives) are not employed, making the process safeand straightforward. A 2-step purification procedure of mPEG2lys is also presented. It is noteworthy thatby following this method pure mPEG2lys was obtained with 41% isolated yield. The mPEG2lys was furtherconverted to the N-hydroxysuccinimidyl ester by diimide activation and successfully used in the pegyla-tion of interferon a-2a.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The covalent attachment of polyethylene glycol (PEG) chainshas been widely used to improve the biopharmaceutical propertiesof several bioactive drugs [1–3]. In this regard, the use of branchedPEG structures is particularly attractive due to the numerousadvantages that branched PEG conjugates offer over the equivalentlinear forms [4]. Among the plethora of available pegylating re-agents, the branched methoxy-PEG having a lysine core (mPEG2lys,compound 1, Fig. 1) has gained considerable attention as activat-able precursor for pegylation [5–14]. In fact, the Y-shapedmPEG2lys 1 is capable of providing a high degree of shielding effec-tiveness. Branched mPEG 1 is not reactive enough to be used di-rectly for conjugation to biomaterials. Therefore, it is usuallyconverted to the more reactive N-hydroxysuccinimidyl ester bymeans of diimide activation [15,16].

Few methodologies for the synthesis of 1 have been publishedor patented [5,17–20]. A simple inspection of the l-lysine structuresuggests that the amino group at the a position might be less reac-tive than the amino group at the e position (the pKa of the e-aminogroup is 1–2 pH units higher than the a-amino group). Hence, asufficiently strong activated mPEG derivative should be used toaccomplish the required urethane bond formation at the less reac-

ll rights reserved.

00; fax: +54 342 4511079.González), cqfina@santafe-

(S.E. Vaillard).

tive position of l-lysine. One usual strategy to achieve mPEG activa-tion involves the initial reaction of mPEG with toxic phosgene ortriphosgene to obtain the corresponding alkoxycarbonyl chlorideor triphosgene derivative [18,20]. These compounds are ratherunstable and, instead of being purified, they are treated with activealcohols to yield electrophilic carbonates. Although several alco-hols have been used or suggested in the literature, the use ofN-hydroxysuccinimide to yield activated mPEG 2a (Scheme 1) isby far the most preferred method. After reacting l-lysine ethyl esterwith the electrophilic mPEG derivative to yield compound 1b, theester is in turn converted to the branched mPEG 1 by hydrolysisunder acid or basic conditions. Another activated PEG derivativethat can be used for the preparation of 1 is mPEG 2b (Scheme 1),which is obtained from mPEG and p-nitrophenylchloroformate,the latter being a toxic compound [21]. It is also well known thatmPEG carbonylimidazole 2c (Scheme 1) is a mild pegylating re-agent that has been conjugated to various proteins [22,23].

It has been recently shown that related alkyl-carbamoylimidazo-lium iodides 3 (Scheme 1), which can easily be obtained fromaliphatic amines and carbonylbisimidazole (CDI) by a two-step pro-cedure, are useful intermediates for the preparation of ureas, amidesand carbamates, among other compounds [24]. Moreover, it hasbeen reported that alkyl-carbamoylimidazolium iodides are roughly300–400 times more reactive than their parent alkyl-carbamoylim-idazoles [24]. Assuming that this reactivity difference might also bemaintained when moving from mPEG-alkoxycarbonylimidazole 2cto the new mPEG-alkoxycarbonylimidazolium iodide 2d (Scheme1), it seemed plausible that the mPEG-imidazolium salt 2d would

Fig. 1. Structure of mPEG2lys 1.

Scheme 2. New synthesis strategy for the preparation of mPEG2lys 1.

108 M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113

be reactive enough to afford the two required urethane bonds pres-ent in mPEG2lys 1. With this idea in hand and challenged by thedisadvantages found in the methods currently available for the syn-thesis of mPEG2lys 1, we developed the new synthetic proceduredepicted in Scheme 2. The proposed synthetic sequence to branchedmPEG2lys 1 involves only three mild reaction steps on which thenew PEG derivative 2d proved to be a useful key intermediate. Asa distinctive feature, by this synthetic route, the use of toxic and dan-gerous phosgene or triphosgene derivatives is avoided. To completethe preparation method of mPEG2Lys 1, a convenient two-step puri-fication procedure is presented. Finally, the purified mPEG2lys 1 wasactivated to N-hydroxysuccinimide ester and the pegylation ofinterferon a-2a was successfully achieved.

2. Experimental

2.1. Materials

mPEG 20 kDa was purchased to JenKem Technology (Allem, USA).N,N0-carbonylbisimidazole (CDI), l-lysine, N-hydroxysuccinimide,N,N-diisopropylethyl amine, N,O-bis(trimethylsilyl) acetamide, andN,N0-dicyclohexylcarbodiimide were supplied by Sigma Aldrich (St.Louis, MO). Acetonitrile was distilled from anhydrous MgSO4 andstored over molecular sieves (4 Å). THF and diethyl ether were freshlydistilled from sodium benzophenone ketyl. DMSO was distilled un-der vacuum and stored over molecular sieves (4 Å). Dry dichloro-methane was obtained by distillation from P2O5. Methyl iodide wasprepared according to a reported method [25]. All other reagentsand solvents were used as received from the suppliers. Interferona-2a was obtained from Pablo Cassará Laboratory (Buenos Aires,Argentina). Vivaflow 200 ultrafiltration cassettes were purchasedfrom Sartorius (Hannover, Germany). XK 50/20 and XK 26/20 col-umns, and Phenyl Sepharose HP were obtained from GE Healthcare(Piscataway, NJ). Fractogel EMD COO� (M) was supplied by MerckChemicals (Darmstadt, Germany).

2.2. Physico-chemical characterization and analyses

1H NMR experiments were performed in CDCl3 in a BrukerAvance 300 MHz spectrometer and referenced to the residual sol-vent signal. A Bruker Biflex MALDI TOF mass spectrometer was usedto determine molecular weights of different mPEG fractions. UV–Visanalyses were performed on a Shimadzu UV–Vis spectrophotome-ter. SE-HPLC (size exclusion-HPLC) analyses were performed in aShimadzu apparatus equipped with a TSK Gel 3000SW, 7.5 �600 mm column (Tosoh) with UV detection at 280 nm. The mobilephase for the analyses of different species in the conjugationreaction and the chromatographic purification of the conjugatewas 50 mM sodium acetate buffer pH = 5.2, 0.2 M NaCl, 10% ethanol.

Scheme 1. Key step reaction for the synthe

Polyacrilamide gel electrophoresis (SDS–PAGE) was performedaccording to the methods of Laemmli [26], with 8% of polyacrila-mide, to evaluate PEG species in crude reaction mixtures. Afterrunning, gels were rinsed with distilled water and placed in a 5%barium chloride solution. The gels were maintained for 10 minwith gentle mixing, and then they were rinsed again, and placedin a 0.1 N iodine solution for 5–10 min. In the case of proteins orPEG-protein conjugates, 15% polyacrilamide gels were run, andstandard procedures with Coomasie brilliant blue were followedto visualize them.

2.3. Synthesis and purification

2.3.1. Synthesis of mPEG-carbonylimidazole (2c)mPEG-OH 20 kDa (0.1 mmol) was dissolved in anhydrous THF

(6 mL) at 60 �C. CDI (0.3 mmol) was added and the solution wasstirred at 60 �C for 18 h. The solvent was removed under vacuum.The residue was dissolved in water and then 5-fold extracted withchloroform (25 mL each). The organic phase was evaporated at re-duced pressure and dried (5 mm Hg) until constant weight. Awhite solid was obtained (1.98 g, 99%). 1H NMR (300 MHz – Cl3CD),d: 3.35 (s, 3H, OMe); 3.60 (brs, mPEG chain); 4.43–4.52 (m, super-imposed with mPEG chain, CH2OC(O)); 7.04 (s, 1H, Im-H); 7.40 (s,1H, Im-H); 8.11 (s, 1H, Im-H) (see Fig. 2, in Section 3).

2.3.2. Synthesis of mPEG-carbamoylimidazolium salt (2d)1.98 g of product 2c was dissolved in 4 mL of acetonitrile at

room temperature. Freshly prepared methyl iodide was added(1.0 mmol), and the solution was stirred at room temperature for16 h. The solvent was removed under reduced pressure and theresulting solid residue was dried (5 mm Hg) until constant weight.A yellow solid was obtained (1.94 g, 98%). 1H NMR (300 MHz –Cl3CD), d: 3.36 (s, 3H, OMe); 3.63 (brs, mPEG chain); 3.86 (m,superimposed with mPEG chain peak, CH2OC(O)); 4.06 (s, 3H,CH3); 7.51 (s, 2H, 2� Im-H); 9,96 (s, 1H, Im-H) (see Figure, inSection 3).

2.3.3. Synthesis of mPEG2lys (1)2.3.3.1. Preparation of Me3SiNH(CH2)4(COOSiMe3)-NHSiMe3 solution(4). A mixture of l-lysine (0.5 mmol), N,O-bis(trimethylsilyl)-acet-amide (26.2 mol) and acetonitrile (0.30 mL) was irradiated in an

sis of 1 with different activated mPEGs.

Fig. 2. 1H NMR spectrum of 2c.

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113 109

ultrasound sonicator at room temperature until complete dissolu-tion of the reagents (5 min).

2.3.3.2. Synthesis of mPEG2lys (1). mPEG-alkoxycarbonylimidazoli-um iodide 2d (1.94 g) was dissolved in 8 mL of 1:1 acetoni-trile:DMSO mixture. 81 lL of the solution obtained previouslyand N,N-diisopropylethylamine (0.18 mmol) were added. The reac-tion mixture was stirred at 85 �C overnight and allowed reachingroom temperature. Brine solution was added and the aqueousphase was 5-fold extracted with dichloromethane (20 mL each).The combined organic extract was evaporated and dried at reducedpressure (5 mm Hg) until constant weight. A white solid was ob-tained (1.90 g, 98%). SDS–PAGE analyses of the crude reaction mix-ture indicated the formation of mPEG2lys 1 (see Fig. 5, lane 4 inSection 3). Under identical reaction conditions, but using alkoxy-carbonylimidazole 2c instead of alkoxycarbonylimidazolium io-dide 2d, product 1 was not obtained, as shown by SDS–PAGEanalysis of the crude reaction mixture (Fig. 5, lane 3).

2.3.4. Purification of mPEG2Lys (1)2.3.4.1. Pre-purification step. An aqueous solution containing 1.90 gof crude mPEG2lys 1 was diafiltered through a 50,000 MW cutoffPES membrane using a 0.2 M NaCl solution, at a flow rate of120 L m�2 h�1. After one ultrafiltration cycle with 4 L of 0.2 M NaCl,the solution was 5-fold extracted with dichloromethane. The com-bined organic extract was evaporated and dried under vacuum(5 mm Hg) until constant weight (1.425 g, 75%).

2.3.4.2. Purification of 1 by hydrophobic interaction chromatogra-phy. 1.425 g of the pre-purified sample of 1 was dissolved in 4 MNaCl solution (concentration of 15 mg/mL), and loaded on XK 50/20 column packed with Phenyl Sepharose HP (GE Healthcare)(CV = 250 mL). The purification process was conducted under con-ditions of stepwise gradient elution by ionic strength reduction.Eluting samples containing pure mPEG2lys were identified bySDS–PAGE analysis, pooled and 4-fold extracted with dichloro-methane (30 mL each). The combined extract was evaporatedand dried at reduced pressure (5 mm Hg). The solid obtained

(0.826 g, 41.3% isolated yield starting from mPEG 20 kDa) was ana-lyzed by SDS–PAGE (BaCl2/I stain, Fig. 5) RP-HPLC-ELSD and char-acterized by MALDI-TOF, 1H NMR (Figs. 6 and 7 in Section 3). 1HNMR (300 MHz – Cl3CD), d: 0.90–0.95 (m, 2H, lysine skeleton);1.2–1.4 (m, 6H, lysine skeleton); 3.09 (s, 6H, OMe); 4.14 (m, 2H,CH2OC(O)); 7.49 (s, 1H, NH); 7.65 (s,1H, NH) (see Fig. 7 inSection 3).

2.4. Conjugation of IFN a-2a

2.4.1. Activation of mPEG2lys1 to the N-hydroxysuccinimidyl ester (mPEG2lys-OSu): activation

of 1 was accomplished by a slight modification of a known method[6,27] as follows: N-hydroxysuccinimide (0.062 mmol) wasdissolved in 2 mL of anhydrous dichloromethane and 2 mL ofanhydrous THF under nitrogen atmosphere and was kept understirring in an ice bath. mPEG2lys (0.0206 mmol) and dicyclohexyl-carbodiimide (0.041 mmol) were added under nitrogen. The solu-tion was stirred for 2 h in the ice bath. A new aliquot ofdicyclohexylcarbodiimide (0.02 mmol) was then added and thereaction mixture was stirred for 30 min in the ice bath and thenmaintained for 16 h at 4 �C without stirring. Dry diethyl etherwas added to the reaction tube (30 mL), and the precipitated prod-uct was separated by centrifugation. The solid was washed withdry diethyl ether (30 mL), dried and re-dissolved in 2 mL of aceto-nitrile. Acetic acid (35 lL) was added and the solution was kept 1 hunder stirring at room temperature. The upper phase was sepa-rated by centrifugation and the product was precipitated withdry diethyl ether, separated by centrifugation and once morewashed and precipitated. The obtained solid was dried at reducedpressure (5 mm Hg) until a constant weight (0.735 g, 89%). Theactivation was achieved in 98% yield, as shown by a known spec-trophotometric assay [28].

2.4.2. Conjugation of (mPEG2lys-OSu) with IFN a-2amPEG2lys-OSu (250 mg) was dissolved in 2.3 mL of 1 mM HCl

previously cooled to 4 �C, and stirred in an ice bath. When the re-agent was dissolved IFN a-2a (45 mg, 3.25 mg/mL, 50 mM sodium

110 M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113

borate buffer pH 8.0) was quickly added, and the reaction allowedto proceed for 3 h at 4 �C under gentle stirring [6,27]. The solutionwas quenched by the addition of acetic acid to pH 4.5, then dilutedeight times with 10 mM ammonium acetate pH 4.5 and loaded onan ion exchange chromatography column (matrix: Fractogel EMDCOO� (M); column: XK 26/20, GE Healthcare; CV = 60 mL; 4 �C).The purification process was conducted under conditions ofstepwise gradient elution with the following buffers: 40 mMammonium acetate pH 4.5; 0.12 M NaCl in 40 mM ammoniumacetate pH 4.5; 0.5 M NaCl in 40 mM ammonium acetate pH 4.5and 1 M NaCl in 40 mM ammonium acetate pH 4.5. Eluting sam-ples were monitored by UV absorbance at 280 nm. Fractions con-taining conjugate IFN a-2a – mPEG2lys were pooled andconcentrated using Amicon� Ultra centrifugal filters (regeneratedcellulose, 10,000 MW CO). SE-HPLC and SDS–PAGE gels (stainedwith Coomasie brilliant blue, and BaCl2/I; using a commerciallyavailable Ref. [29]) were used to evaluate the conjugation reactionand the chromatographic purification (Fig. 8 in Section 3). Concen-tration of proteins was determined by UV absorbance at 280 nm,and by Lowry protein assays. The isolated final yield of the conju-gation procedure was 26%.

3. Results and discussion

3.1. Preparation and purification of mPEG2Lys 1

The complete process for the synthesis of branched mPEG2lys 1is depicted in Scheme 2. The strategy involves only few steps usuallyunder mild reaction conditions. Noteworthy, none of these stepsrequire inert atmosphere or special experimental setup. Given thatcompound 1 bearing two 20 kDa mPEG chains is one of the mostuseful branched mPEGs for the pegylation of bioactive molecules,we decided to explore the envisaged strategy using mPEG 20 kDaas starting material. The first step of the synthesis strategy involvesthe well-known reaction of mPEG with CDI to afford mPEG-alkoxi-carbonylimidazole 2c under mild reaction conditions (Scheme 2).Other similar methods [22,23] for the preparation of activated poly-mer 2c have also been published. The 1H NMR analysis of the crude2c clearly indicated the formation of the product, and the ratio ofOCH3 to the 3 � 1 H imidazole signals suggested the reaction pro-ceeded to completion; hence the crude product was used in the nextstep without further purification (Fig. 2).

Looze and coworkers determined a e value of 3590 M�1 cm�1

for t-butyloxycarbonylimidazole (water, 231 nm, 25 �C), and itwas indicated that this value can be used with confidence for com-pound 2c with a mPEG chain of 5 kDa [23]. The UV–Vis spectrum of

Fig. 3. (a) UV–Vis spectrum of 2c a

compound 2c is presented in Fig. 3a. This spectrum shows a peak atkmax at 228 nm, in good agreement with published data. Moreover,we determined a e value for compound 2c of 3760 M�1 cm�1

(water, 231 nm, 25 �C) suggesting that the reaction of CDI withmPEG (20 KDa) was complete.

After the seminal work of Salvensen and Rapoport, carbamoylim-idazolium salts have received little attention in organic synthesis.However, in last few years Batey has shown that carbamoylimidazo-lium iodides, which can easily be obtained from amines and CDI fol-lowed by alkylation with methyl iodide, are useful intermediates forthe preparation of ureas among other compounds [24,30,31]. Withthe aim of taking advantage of the aforementioned reactivity differ-ence between carbamoylimdazoles and carbamoylimidazolium io-dides, compound 2c was reacted with methyl iodide underextremely mild conditions to afford alkoxycarbonylimidazoliumiodide 2d with good yield (98%, room temperature, 16 h) [24]. Afterremoval of the reaction solvent and volatile products, the 1H NMRanalysis of the crude product (Fig. 4) indicated the formation ofthe product, and the ratio of Im-CH3 to the OCH3 and 3 � 1 H imid-azole signals suggested that the akylation reaction was complete in16 h at room temperature (Scheme 2).

Imidazolium salt 2d is not stable in aqueous solutions. TheUV–Vis spectrum of compound 2d is presented in Fig. 3b. Compound2d presents peaks at kmax at 360 and 291 nm (acetonitrile, 25 �C).The estimated e values for these peaks are 1907 and 3808 M�1 cm�1,respectively.

The conversion to the TMS derivatives is a well-establishedmethodology for the derivatization of amino acids. Under ultra-sound irradiation, l-lysine was converted to the TMS derivative 4using excess of N,O-bis(trimethylsilyl)acetamide and acetonitrileas solvent (Scheme 2) [32]. Compound 4 is completely soluble inorganic solvents making unnecessary the use l-lysine ethyl ester.It addition, since the TMS group can easily be removed by standardaqueous work-up, an additional hydrolysis step after the couplingof the activated mPEG reagent should not be needed.

We found that heating a mixture of 2d and 4 at 85 �C for 16 h in1:1 acetonitrile:DMSO using diisopropylethylamine as base, fol-lowed by standard aqueous work-up, constituted the optimal reac-tion conditions for the synthesis of mPEG2lys 1. It is noteworthythat only 10 mol% (5 eq.%) were enough to obtain mPEG2lys withgood yield. The SDS–PAGE analysis of the crude product showedthe required branched structure 1 (40 kDa) and unreacted mPEGor monomeric mPEGlys (both 20 kDa) in an estimated ratio of60:40 (by visual inspection of SDS–PAGE; see Fig. 5, lane 4). As re-ported for PEG-proteins conjugates [6,27,33] bearing this kind ofbranched PEG structure, the electrophoretic mobility is consider-

nd (b) UV–Vis spectrum of 2d.

Fig. 4. 1H NMR spectrum of 2d.

Fig. 5. SDS–PAGE analysis from different samples: lane 1: protein molecular weightmarkers; lane 2: mPEG (20 kDa); lane 3: crude product mixture of the reaction ofmPEG derivative 2c with 4; lane 4: crude product mixture of the reaction of mPEGderivative 2d with 4: lane 5: pre-purified sample of 1 after being diafiltered; lane 6:purified mPEG2lys by HIC.

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113 111

ably slowed owing to the large hydrodynamic volume of PEG (eachethylene oxide subunit of PEG can form an adduct with 3 mole-cules of water) [34]. As shown in Fig. 5, in the electrophoresis pro-cedures, branched mPEG2lys run as a globular protein of 75 kDa. Inagreement with its expected lower reactivity, mPEG-alkoxycarb-onylimidazole 2c reacted with 4 affording 1 in trace amounts, asshown by the SDS PAGE analysis (Fig. 5, lane 3).

The development of adequate techniques for the purificationand characterization of PEGs and PEG derivatives has been a criti-cal issue for the progress on the synthesis of new and interestinglinear and branched PEGs. Given the fact that PEG is UV invisibleand that, in high molecular weight PEGs, the changes introducedupon modification of the reactive hydroxyl end group are almostundetectable when compared with polyethylene glycol chain,more sophisticated methods are required to separate them.

Branched mPEG2lys 1 has been separated from the monomericforms (native mPEG or mPEGlys) by gel permeation and ion ex-change chromatography [5]. Although being useful, limitedamounts of mPEG2lys were processed in gel permeation purifica-

tion and large column volumes were employed in ion exchangechromatography. Ultrafiltration methods have been specially stud-ied to separate pegylated proteins from unreacted materials basedon differences in their size [35–38]. It has been reported that thenumber, length and structure of the polymer chain attached toproteins influence the sieving coefficients, not allowing their esti-mation with only the hydrodynamic volume [38]. Moreover, athigh filtrate flow rates an elongation on the PEG chain is produced,which increases when longer PEG chains are attached [39].

With the aim of using chromatographic techniques withoutcompromising the capacity of the matrix, we performed a pre-puri-fication of mPEG2lys 1 by economic and robust tangential flowultrafiltration in a diafiltration mode (PES membrane and50,000 MW cutoff, see Section 2). An 80% enriched mPEG2lys 1sample (estimated by visual inspection of SDS–PAGE gels, seeFig. 5, lane 5) was purified by HIC, eluting mPEG2lys 1 in the pureform. The product was characterized by 1H NMR, MALDI-TOF andRP-HPLC-ELSD (Figs. 6 and 7) [5]. To the best of our knowledge,HIC has not previously been employed to separate mPEG2lys fromunreacted PEG. We found that this procedure is quite successful forthis purpose being able to efficiently separate with high resolutionmore than 2 g of pre-purified sample using a column volume of250 mL.

The synthesis strategy and the purification procedure describedherein allowed us to obtain pure mPEG2lys 1 with 41% isolatedyield. Unfortunately, we have not been able to properly comparethis overall yield with those obtained in previously publishedmethods because the synthesis and purification procedures forpreparing this branched PEG derivative have been described in de-tail, but the final yields of the procedures have not been clearlyreported.

3.2. Activation of mPEG2lys and pegylation of IFN a-2a

With the aim of evaluating the performance of mPEG2lys 1 ob-tained as previously indicated, branched mPEG 1 was converted tothe N-hydroxysuccinimidyl ester (mPEG2lys-OSu) under reactionconditions similar to those indicated in the literature (diimide acti-

Fig. 6. (a) MALDI-TOF spectrum of pre-purified crude mixture by diafiltration; peaks of 20 kDa PEGs species, mPEG2lys (40 kDa) and oligomer structures can be seen. (b) RP-HPLC-ELSD chromatogram of the same sample.

Fig. 8. (a) Chromatogram of SE-HPLC analysis of crude pegylation reaction, showing the presence of the different species, (b) Coomasie brilliant blue stained gel and (c)barium/iodine stained gel to evaluate pegylation reaction. Lane 1: protein molecular weight markers; lane 2: IFN a-2a; lane 3: PEGASYS�; lane 4: crude pegylation reaction;lane 5: purified mPEG2lys-IFN a-2a.

Fig. 7. 1H NMR spectrum of 1.

112 M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113

M. González et al. / Reactive & Functional Polymers 72 (2012) 107–113 113

vation at 0 �C) [6,27]. Using a reported assay, the yield of theactivation reaction was 98% [28].

IFN a-2a was pegylated with mPEG2lys-OSu under known reac-tion conditions (sodium borate buffer, pH 8, at 4 �C using 5-foldexcess of mPEG2lys-OSu) [6,27]. SE-HPLC analysis of the crudepegylation reaction indicated 35% of monopegylated protein, 2.5%of oligomer species, and 62.5% of unmodified interferon (seeFig. 8a) using commercial mPEG2lys-IFN a-2a as reference.

After quenching, the crude pegylation reaction was diluted andimmediately loaded to Fractogel EMD COO� (M) column for purifi-cation purposes (see Section 2 for details). SDS–PAGE analyses(stained with Coomasie brilliant blue, and BaCl2/I; using a com-mercial conjugate reference) were used in the different stages ofthe process to confirm the presence of the conjugate, and toevaluate the chromatographic purification (Fig. 8b and c). Concen-tration of proteins was determined by UV absorbance at 280 nm,and by Lowry protein assay. Similar to other reported methodolo-gies, the chromatographic yield was 72%, and the conjugatemPEG2lys-IFN a-2a was obtained in a 26% isolated yield.

4. Conclusions

mPEG2lys 1 has been one of the most studied branched PEGs forthe pegylation of different active biomolecules. Despite its provenutility, few methods for its synthesis have been reported. Takingadvantage of the increased reactivity of mPEG-alkoxycarbonylimi-dazolium iodide 2d, compared with that of the parent alkoxycarb-onylimidazole 2c, a new synthesis strategy and a purificationprotocol for the preparation of mPEG2lys 1 (40 kDa) were devel-oped (41% isolated yield). The synthetic method involves onlythree reaction steps under mild conditions, using standard chemi-cals and experimental set ups. Moreover, the use of toxic or dan-gerous reagents, such as phosgene or triphosgene is avoided. Inthis regard, the key functionalized mPEG 2d proved to be a usefulalternative to the well-known mPEG-N-hydroxysuccinimidyl or p-nitrophenyl carbonates. Furthermore, the use of a silylated form ofl-lysine suppresses the need for the standard hydrolysis step aftercoupling of the mPEG chains, as is usually required with l-lysineethyl ester.

The two-step purification procedure described before involvesthe initial enrichment of the required product by a simple ultrafil-tration process that would increase the life of the chromatographicmatrix (HIC), at the same time that it allows the purification of 2 gof enriched mPEG2lys per run under standard scale set up and withexcellent resolution.

The performance of mPEG2lys 1 was evaluated by its conversionto mPEG2lys-OSu by diimide activation and by the successful pegy-lation of IFN a-2a using this reactive ester.

Acknowledgments

The authors wish to express their gratitude to Agencia Nacionalde Promoción Científica y Tecnológica (ANPCyT), to Consejo Nacion-al de Investigaciones Científicas y Técnicas (CONICET), to Universi-dad Nacional del Litoral (UNL) of Argentina and to CARBONFE, forthe financial support given to this contribution. M. González deeply

acknowledges having been granted a fellowship from CONICET-CARBONFE.

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